Several independent problems will be pursued under this grant. (l) Homogeneous histidine decarboxylase from Lactobacillus 30a contains covalently bound pyruvate and a functionally essential -SH group near the active center. Sequence studies are planned to determine the proximity of these two groups in the primary structure; if suitable crystals can be obtained we will explore collaborative determination of the 3-dimensional structure of the enzyme. Histidine methyl ester inhibits irreversibly by binding at the active site. If partial hydrolysis products can be obtained, further mapping of the active site will be possible. Histidine decarboxylase arises from an inactive proenzyme that lacks the pyruvate residue and contains only one species of peptide chain. Conversion of proenzyme to active enzyme involves chain cleavage and conversion of a serine residue to a pyruvate residue. We will determine the effect of inhibitors, ionic strength, crude extracts, etc., on this conversion to determine its precise nature. (2) Studies of active site residues in tryptophanase and D-serine dehydratase from E. coli will be continued in an attempt to further elucidate the mechanism of these reactions. (3) We will continue studies, already begun, of the uptake and transport of vitamin B6 and pantothenic acid by both E. coli and organisms that are auxotrophic for these vitamins. (4) The terminal phases of vitamin B6 utilization (as a sole carbon source for bacteria) involve a flavin-dependent oxygenase that reduces the substrate in one position while inserting oxygen at another. Isolation of the crystalline enzyme, redetermination of the extent of O2-incorporation, and its iron content should partly clarify the nature and mechanism of action of this unusual oxygenase.